重组福安泰-03融合蛋白表达条件的优化及其生物学活性检测  

Optimization of Expression Conditions of Recombinant Fuantai-03 and Detection of its Biological Activities

作　　者：张永平[1] 马润娣[1] 于立坚[1] 苏伟明[1] 廖铭能[1] 黄来珍[1] 于廷曦[1,2] 

机构地区：[1]广东海洋大学海洋药物研究与开发重点实验室,湛江524025 [2]Cell Biology Group,Department of Surgery,Department of Pathology,University of Maryland School of Medicine and Baltimore Veterans Affairs Medical Center,Baltimore,MD 21201,USA

出　　处：《生物医学工程学杂志》2012年第5期923-928,共6页Journal of Biomedical Engineering

基　　金：国家科技部863计划海洋技术领域专题基金资助项目(2007AA092422);国家自然科学基金资助项目(30271493);广东省自然科学基金重点项目资助(021386);广东省海洋与渔业局科技兴海重大项目资助(A200099B01)

摘　　要：福安泰-03(FAT-03)是从赤魟组织中分离得到,具有强抗血管生成活性的蛋白。本研究通过DNA重组技术获得了重组福安泰-03(rFAT-03)融合蛋白,运用响应面试验设计法优化表达条件,采用GST亲和柱分离纯化表达蛋白,比较分析不同培养时间、诱导剂浓度、诱导温度和诱导时间对可溶性融合蛋白表达量的影响。结果表明,当培养时间为6.13h、诱导温度为19.71℃、诱导剂(IPTG)浓度为0.36mmol/L、诱导时间为13.60h条件下可获得较好的表达效果,可溶性目的蛋白GST-rFAT-03的得率为7.57mg/L。对获得的rFAT-03生物活性检测显示,rFAT-03能明显抑制鸡胚绒毛尿囊膜血管生成,且其效果与剂量相关。为后续对该蛋白的分离纯化和生物学功能研究奠定了基础。Fuantai03（FAT03）, isolated from the Dasyatis akajei, has a strong antiangiogenic actiity. The recombi nant Fuantai03 （GST/rFAT03）fusion protein can be obtained with the DNA recombination technology. In this study, expression conditions of GST/rFAT03 were optimized by response surface experimental design method. The constructed engineering bacteria containing GST/rFAT03 plasmid was induced by isopropy13Dthiogalactosid （IPTG）, the GST affinity column was used for isolation and purification, and then the effects of different culture time, IPTG concentration, induction temperature and induction time on the amount of soluble GST/rFAT03 fusion protein were compared. The culture time for optimal expression was 6.13 h, IPTG concentration was 0. 36 mmol/L, induction temperature was 19.71℃, and induction time was 13.60 h. The amount of soluble GST/rFAT03 fusion protein was 7.57 mg/L under above mentioned expression conditions. The results also showed that rFAT03 signifi cantly inhibited angiogenesis in chicken chorioallantoic membrane in a dosedependent manner. Moreover, the soluble form of the target protein is useful for further work on purification and on studying its biological function.

关 键 词：GST/rFAT-03融合蛋白 可溶性融合蛋白 响应面设计法 表达条件 优化 生物活性 

分 类 号：Q786[生物学—分子生物学]